This invention relates to a method for the determination of lactate and lactate dehydrogenase.
Lactate dehydrogenase (LDH) is a dehydrogenase enzyme which catalyzes the oxidation of L-lactate to pyruvate through a nicotinamide adenine dinucleotide (NAD) coupled enzyme reaction as follows: EQU lactate + NAD.sup.+ .sup.LDH .revreaction. pyruvate + NADH + H.sup.+
The determination of LDH is of clinical significance in the diagnosis of various diseases. Thus, an elevated level of LDH is indicative of a myocardial infarction. Elevations of serum LDH are also observed in liver disease and in patients with certain types of carcinomas.
Numerous methods have been developed heretofore for the determination of LDH, including a variety of optical tests based on either the forward or reverse of the foregoing reaction, namely the oxidation of lactate to pyruvate or the reduction of pyruvate to lactate. The pH optimum of the forward reaction is 8.8 to 9.8, whereas for the reverse reaction it is 7.4 to 7.9. In the forward reaction the rate of increase, and in the reverse reaction the rate of decrease, in absorbance at 340 nm serves as the parameter of the enzyme activity in various spectrophotometric determination methods. The method which employs the forward reaction has been recommended as providing greater stability. N. Eng. J. Med. 261, 1259-66 (1959) and Clin. Chem. 9, 391-9 (1963).
Several colorimetric methods for the determination of LDH also are based on the foregoing coupled enzyme reaction. In one method, the pyruvate is reacted with 2,4-dinitrophenylhydrazine in a terminal indicator reaction to form the characteristic brown color of the corresponding 2,4-dinitrophenylhydrazone pyruvate which is measured colorimetrically at 440 nm.
In another colorimetric procedure, a terminal acceptor dye which gives the color change and an intermediary electron transfer agent to transfer hydrogen from the substrate to NAD are both employed. Thus, a phenazine methosulfate carrier has been used to couple the electron transfer between NADH and the terminal acceptor dye 2,6-dichlorophenol-indolphenol as described by Ells, Arch. Biochem. Biophys. 85, 561-62 (1959). Meldola blue is another typical electron carrier used heretofor in a similar such colorimetric determination of LDH as can be seen from U.S. Pat. No. 3,732,147.
While the foregoing optical methods for the determination of LDH are useful, they have the disadvantage in that each individual serum sample assay takes at least about 5 to 10 minutes to complete.